WO2016031869A1 - スチルベン化合物を有効成分とする角結膜疾患又は老視の予防及び/又は治療のための医薬組成物 - Google Patents
スチルベン化合物を有効成分とする角結膜疾患又は老視の予防及び/又は治療のための医薬組成物 Download PDFInfo
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- ATYQGOFMEQUNMJ-BUHFOSPRSA-N C/C(/c1ccccc1)=C(/C)\c1ccccc1 Chemical compound C/C(/c1ccccc1)=C(/C)\c1ccccc1 ATYQGOFMEQUNMJ-BUHFOSPRSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/05—Phenols
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/04—Artificial tears; Irrigation solutions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/10—Ophthalmic agents for accommodation disorders, e.g. myopia
Definitions
- the present invention relates to a pharmaceutical composition for preventing and / or treating keratoconjunctival disease or presbyopia containing a stilbene compound.
- Resveratrol (Resveratrol, 2-trans-1,2- (3,4 ', 5-trihydroxydiphenyl) ethylene) is a known still for its life-longening effect, prevention of cardiovascular-related diseases, and improvement of brain function. It is a kind of benoid polyphenol, and recently, an association with eye diseases has been reported.
- Patent Document 1 reports that transstilbene derivatives such as resveratrol or piceatannol as sirtuin activators are effective as therapeutic agents for various eye diseases such as eye strain.
- the present inventors have found that a derivative in which two methyl groups are introduced into a vinyl group of a stilbene skeleton stabilizes the activity of such a derivative, and keratoconjunctiva including dry eye.
- the present inventors have found an unexpected effect that is effective in the prevention and / or treatment of diseases and presbyopia (presbyopia), and have completed the present invention.
- such a novel stilbene derivative exhibits a strong antioxidant action as compared with resveratrol.
- Item 1 A pharmaceutical composition for preventing and / or treating keratoconjunctival disease or presbyopia comprising a compound represented by formula (I), a salt thereof or a prodrug as an active ingredient.
- Item 2 The pharmaceutical composition according to Item 1, wherein m and n are each an integer of 1 to 3. Item 3. Item 3. The pharmaceutical composition according to Item 2, wherein the compound is a compound represented by formula (II), a salt thereof or a prodrug.
- Item 4 The pharmaceutical composition according to Item 3, wherein the compound is a compound represented by formula (III), a salt thereof, or a prodrug.
- a prophylactic and / or therapeutic agent for keratoconjunctival disease or presbyopia comprising a compound represented by formula (I), a salt thereof or a prodrug as an active ingredient.
- n and n are each an integer from 0 to 5, m + n ⁇ 1, and each aromatic ring of the compound represented by the formula (I) may be substituted.
- Item 6 A compound represented by the formula (I), a salt or a prodrug thereof, which is used for preventing and / or treating keratoconjunctival disease or presbyopia.
- Item 7 A method for preventing and / or treating keratoconjunctival disease or presbyopia comprising administering to a patient an effective amount of a compound represented by formula (I), a salt or a prodrug thereof.
- the compound of the present invention or a salt thereof is useful for the prevention and / or treatment of keratoconjunctival disease or presbyopia.
- the compound of the present invention or a salt thereof is also useful as an antioxidant.
- the graph which shows the result of an electron spin resonance (ESR) measurement.
- A Amount of tyrosine radical
- B Amount of hydroxy radical.
- 5,50,500 are 5 ⁇ M, 50 ⁇ M, and 500 ⁇ M, respectively
- R, DR, and DHR are resveratrol, dimethylresveratrol (formula (V)), and dimethylhydroxyresveratrol (formula (VI)), respectively.
- Show. Std standard substance.
- the graph which shows the body weight of the rat of a non-smoking process group and a smoking process group.
- the graph which shows the average amount of tears of 5-6 and 10-11 minutes after drug administration when various drugs are administered after smoking treatment.
- Nonsmoking no smoking treatment, Smoking: smoking treatment, S + R: smoking treatment and resveratrol administration, S + DR: smoking treatment and dimethylresveratrol administration, S + DHR: smoking treatment and dimethylhydroxyresveratrol administration.
- the graph which shows the fluorescence-staining score at the time of administering various chemical
- the graph which shows the elasticity modulus of the lens at the time of administering various chemical
- the graph which shows the average amount of tears of 5-6 and 10-11 minutes after drug administration when various drugs are administered after smoking treatment.
- Nonsmoking no smoking treatment, Smoking: smoking treatment, DR: smoking treatment and dimethylresveratrol administration, Diquafosol: diquafosol sodium administration.
- the graph which shows the fluorescence-staining score at the time of administering various chemical
- treatment means cure or amelioration of a disease or symptom, or suppression of a symptom, and includes “prevention”.
- Prevention means preventing the onset of a disease or symptom.
- the compounds of the present invention include compounds represented by the following formula (I).
- n and n are each an integer from 0 to 5, and m + n ⁇ 1.
- 4- [3- (4-hydroxyphenyl) but-2-en-2-yl] benzene-1,2-diol represented by the following chemical formula (i) is excluded from the compound of the present invention.
- Each aromatic ring of the compound represented by the formula (I) is a lower alkyl group having 1 to 6 carbon atoms (for example, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, se c-butyl, t e r t- Butyl, cyclopropyl, cyclobutyl, etc.), lower alkoxy groups having 1 to 6 carbon atoms (eg, methoxy, ethoxy, propoxy, isopropoxy, etc.), halogen atoms (eg, fluorine, chlorine, bromine, etc.), etc. Good.
- the compound represented by the above formula (I) there are a trans isomer (E) and a cis isomer (Z) with respect to the vinyl group of the stilbene skeleton. A mixture thereof may also be used.
- the compound represented by formula (I) is a trans isomer.
- the compounds of the present invention include both trans and cis forms.
- the specific compound of the compound represented by the formula (I) may exist in a stereoisomeric form, but in the present invention, cis and trans isomers, R- and S-enantiomers, diastereomers All such compounds are included, including the (D) -isomer, (L) -isomer, racemic mixtures thereof, and other mixtures.
- asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers and mixtures thereof are encompassed by the compounds of the present invention.
- the position of the hydroxyl group on the aromatic ring may be any of the ortho, meta, and para positions, and may have activity related to keratoconjunctival disease or presbyopia or antioxidant regardless of these positions. It has been found by our previous research.
- n and n are each an integer from 1 to 3. In another embodiment, m and n are each an integer from 1 to 3, and the total number of hydroxyl groups on the aromatic ring is 3-6.
- the compound of the present invention is a compound represented by the following formula (II).
- n 1 and n 1 are each an integer from 0 to 2.
- the compound of the present invention is a compound represented by the formula (III).
- n 2 is an integer from 0 to 2.
- the compound of the present invention is a compound represented by the formula (IV).
- n 3 is 0 or 1
- the compound is trans.
- the above-mentioned compound of the present invention is (E) -5- [3- (4-hydroxyphenyl) but-2-en-2-yl] benzene represented by the following formula (V): 1,3-diol or (E) -4- [3- (3,5-dihydroxyphenyl) but-2-en-2-yl] benzene-1,2-diol represented by the formula (VI) is there.
- the compound represented by the formula (I) of the present invention may form a salt, and as the salt, an acid addition salt such as an inorganic acid salt (for example, hydrochloride, sulfate, hydrobromide, Phosphates, etc.), organic acid salts (eg acetate, trifluoroacetate, succinate, maleate, fumarate, propionate, citrate, tartrate, lactate, oxalate, Methanesulfonate, p-toluenesulfonate, and the like).
- an acid addition salt such as an inorganic acid salt (for example, hydrochloride, sulfate, hydrobromide, Phosphates, etc.), organic acid salts (eg acetate, trifluoroacetate, succinate, maleate, fumarate, propionate, citrate, tartrate, lactate, oxalate, Methanesulfonate, p-toluenesulfonate, and the like).
- the present invention includes various hydrates and solvates of the compound represented by the formula (I) or a salt thereof and a polymorphic substance.
- the present invention may be a prodrug of the compound represented by formula (I).
- the prodrug of the compound represented by formula (I) refers to a compound that is converted into the compound represented by formula (I) by a reaction in vivo.
- Examples of the prodrug of the compound represented by the formula (I) include compounds in which the hydroxyl group of the compound represented by the formula (I) is acetylated, acylated, alkylated, phosphorylated, sulfated, or borated. It is done. These compounds can be produced by a method known per se.
- the prodrug of the compound IV represented by the formula (I) may be itself or a pharmacologically acceptable salt.
- a salt include a salt with an inorganic base or an organic base when the prodrug of the compound ⁇ represented by the formula (I) has an acidic group such as a carboxyl group, and is represented by the formula (I).
- the prodrug of the compound IV has a basic group such as an amino group, a salt with an inorganic acid or an organic acid can be mentioned.
- the prodrug of the compound represented by formula (I) may be either a hydrate or a non-hydrate.
- the compound represented by the formula (I) is produced by, for example, the method shown below.
- methylmagnesium bromide (MeMgBr) is added dropwise and stirred to a mixture of tetrabutylammonium chloride (Bu 4 NCl) and diethylene glycol dimethyl ether (diglyme) under an argon atmosphere. After cooling the reaction mixture, a THF solution of the compound of formula (2) is added dropwise and further stirred. The reaction mixture is separated and the organic layer is dried. After distilling off under reduced pressure, the residue is purified by column chromatography to obtain the compound of formula (3).
- MeMgBr methylmagnesium bromide
- Bu 4 NCl tetrabutylammonium chloride
- diglyme diethylene glycol dimethyl ether
- boron tribromide (BBr3) is added dropwise to a dehydrated methylene chloride solution of the compound of formula (6) (mixture of cis isomer and trans isomer) in an argon atmosphere.
- the reaction mixture is stirred and then further stirred.
- the reaction mixture is separated, and the aqueous layer is separated with another solvent.
- the obtained organic layer is dried and then distilled off under reduced pressure.
- the residue is roughly purified by column chromatography to obtain the compound of formula (7) as a mixture.
- the compound of the present invention having a hydroxyl group on two aromatic rings and having two methyl groups introduced into the vinyl group of the stilbene skeleton is produced.
- the compound of the present invention is useful as a prophylactic and / or therapeutic agent for keratoconjunctival disease because it is excellent in the prevention and / or treatment effect of keratoconjunctival disease, and contains the compound of the present invention as an active ingredient.
- a pharmaceutical composition for preventing and / or treating is also within the scope of the present invention.
- the keratoconjunctive disease includes dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, corneal ulcer and the like.
- cornea conjunctiva means the cornea and / or conjunctiva
- keratoconjunctival disease refers to a disease in the cornea and / or conjunctiva.
- Diseases such as dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, or corneal ulcer in the present invention are generally diseases included in keratoconjunctival diseases, but are not necessarily caused by keratoconjunctival diseases. It doesn't have to be a thing. Accordingly, the dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, corneal ulcer and the like in the present invention include those that do not depend on keratoconjunctival disease.
- the compound of the present invention is excellent in the prevention and / or treatment effect of presbyopia, it is useful as an agent for the prevention and / or treatment of presbyopia, and presbyopia containing the compound of the present invention as an active ingredient.
- Pharmaceutical compositions for prevention and / or treatment are also within the scope of the present invention.
- the compound of the present invention is excellent in antioxidant action, it is also useful as an antioxidant.
- the compounds of the present invention can be used for mammals including humans (eg, humans, cows, horses, pigs, monkeys, dogs, cats, mice, rats, rabbits, goats, sheep, etc.), preferably humans. Used for.
- the compound of the present invention when contained in a pharmaceutical composition, it can be combined with a pharmaceutically acceptable carrier as necessary, and various administration forms can be adopted depending on the purpose of prevention or treatment, and the form is particularly limited. Examples thereof include eye drops, oral preparations, injections, suppositories, ointments, patches, and the like, and eye drops are preferred. Each of these dosage forms can be produced by a conventional formulation method known to those skilled in the art.
- the eye drops may be any of aqueous eye drops, non-aqueous eye drops, suspension eye drops, emulsion eye drops, eye ointments and the like.
- Such a preparation is prepared as a composition suitable for the dosage form, if necessary, as a pharmaceutically acceptable carrier, for example, an isotonic agent, a chelating agent, a stabilizer, a pH adjusting agent, a preservative, an antioxidant. , A solubilizing agent, a thickening agent, and the like, and can be produced by a method known to those skilled in the art.
- Isotonic agents include glucose, trehalose, lactose, fructose, mannitol, xylitol, sorbitol and other sugars, glycerin, polyethylene glycol, propylene glycol and other polyhydric alcohols, sodium chloride, potassium chloride, calcium chloride and other inorganic salts
- the blending amount is preferably 0 to 5% by weight based on the total amount of the composition.
- Chelating agents include edetates such as disodium edetate, disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or its salts, sodium hexametaphosphate Citric acid and the like, and the blending amount thereof is preferably 0 to 0.2% by weight based on the total amount of the composition.
- edetates such as disodium edetate, disodium edetate, trisodium edetate, tetrasodium edetate, and calcium edetate, ethylenediaminetetraacetate, nitrilotriacetic acid or its salts, sodium hexametaphosphate Citric acid and the like, and the blending amount thereof is preferably 0 to 0.2% by weight based on the total amount of the composition.
- the stabilizer examples include sodium bisulfite and the like, and the blending amount is preferably 0 to 1% by weight with respect to the total amount of the composition.
- Examples of the pH adjuster include acids such as hydrochloric acid, carbonic acid, acetic acid, and citric acid. Further, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkali metal carbonates or hydrogen carbonates such as sodium carbonate, Examples thereof include alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, bases such as trometamol, and the like, and the blending amount is preferably 0 to 20% by weight based on the total amount of the composition.
- preservatives include sorbic acid, potassium sorbate, methyl paraoxybenzoate, ethyl paraoxybenzoate, paraoxybenzoate propyl, paraoxybenzoate butyl ester, etc., chlorhexidine gluconate, benzalkonium chloride, benzethonium chloride, Examples thereof include quaternary ammonium salts such as cetylpyridinium chloride, alkylpolyaminoethylglycine, chlorobutanol, polyquad, polyhexamethylene biguanide, chlorhexidine and the like.
- the blending amount is 0 to 0.2% by weight based on the total amount of the composition. Is preferred.
- antioxidant examples include dry sodium sulfite, sodium pyrosulfite, concentrated mixed tocopherol and the like, and the blending amount is preferably 0 to 0.4% by weight with respect to the total amount of the composition.
- solubilizers include sodium benzoate, glycerin, polyvinyl pyrrolidone, macrogol, D-mannitol and the like, and the blending amount is preferably 0 to 3% by weight based on the total amount of the composition.
- thickening agent examples include methylcellulose, ethylcellulose, carmellose sodium, xanthan gum, chondroitin sulfate sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, and the like.
- the content is preferably 0 to 70% by weight.
- the desired components described above are dissolved or suspended in an aqueous solvent such as sterilized purified water or physiological saline, or a non-aqueous solvent such as vegetable oil such as cottonseed oil, soybean oil, sesame oil, or peanut oil.
- aqueous solvent such as sterilized purified water or physiological saline
- non-aqueous solvent such as vegetable oil such as cottonseed oil, soybean oil, sesame oil, or peanut oil.
- the osmotic pressure can be adjusted to a predetermined osmotic pressure and subjected to sterilization such as filtration sterilization.
- an ointment base can be included in addition to the various components described above.
- the ointment base is not particularly limited, but is an oily base such as petrolatum, liquid paraffin, or polyethylene; an emulsion base obtained by emulsifying an oil phase and an aqueous phase with a surfactant; hydroxypropyl methylcellulose, carboxymethylcellulose A water-soluble base composed of polyethylene glycol or the like is preferred.
- the dose depends on the patient's weight, age, sex, symptom, dosage form, number of administrations, etc. Although it is different, usually 0.1 to 1000 ⁇ g, preferably 1 to 200 ⁇ g of the compound of the present invention may be administered to an adult once or divided into several times. In this case, 0.01 to 50 mg / mL, preferably 0.1 to 10 mg / mL, may be instilled once to several times a day once to several drops.
- Step A of 1- (3,5-dimethoxyphenyl) -2- (phenylsulfonyl) propan-1-one (1- (3,5-dimethoxyphenyl) -2- (phenylsulfonyl) propan-1-one)
- Ethyl phenyl sulfone (EtSO 2 Ph) (7.72 g, 45.1 mmol) in THF (60 mL) is cooled to ⁇ 80 ° C. and butyl lithium (2.66 mol / L n-hexane (hereinafter simply referred to as “hexane”).
- hexane butyl lithium (2.66 mol / L n-hexane (hereinafter simply referred to as “hexane”). ) Solution, 17.0 mL, 45.1 mmol) was added dropwise and stirred for 40 minutes.
- Step B 2- (3,5-dimethoxyphenyl) -3- (phenylsulfonyl) butan-2-ol (2-) of (3,5-dimethoxyphenyl) -3- (phenylsulfonyl) butan-2-ol) (3) Preparation Methylmagnesium bromide (1.0 mol / L THF solution, 9.0 mL, 9.0 mmol) in a mixture of tetrabutylammonium chloride (167 mg, 0.6 mmol) and diethylene glycol dimethyl ether (1.28 mL, 9.0 mmol) at 0 ° C under argon atmosphere was added dropwise and stirred for 30 minutes.
- Step C (E) -1,3-dimethoxy-5- [3- (phenylsulfonyl) but-2-en-2-yl] benzene ((E) -1,3-dimethoxy-5- [3- (phenylsulfonyl) ) but-2-en-2-yl] benzene) (4) Preparation of Compound 3 (1.71 g, 4.89 mmol) in methylene chloride (40 mL) at 0 ° C. under argon atmosphere.
- the reaction mixture was partitioned between saturated aqueous sodium hydrogen carbonate solution and hexane, and the organic layer was dried over anhydrous sodium sulfate.
- the residue obtained by evaporation under reduced pressure was dissolved in carbon tetrachloride (17 mL), iodine (1.77 g, 6.98 mmol) was added at 0 ° C. under an argon atmosphere, and the mixture was stirred at room temperature for 90 minutes.
- the reaction mixture was partitioned between saturated aqueous sodium hydrogen carbonate solution (100 mL), saturated aqueous sodium thiosulfate solution (100 mL) and methylene chloride, and the organic layer was evaporated under reduced pressure.
- the obtained residue was further separated with a 10% aqueous potassium fluoride solution and methylene chloride, and the organic layer was dried over anhydrous sodium sulfate.
- Step E 1,3-Dimethoxy-5- [3- (4-methoxyphenyl) but-2-en-2-yl] benzene (1,3-dimethoxy-5- [3- (4-methoxyphenyl) but-2 -en-2-yl] benzene) (6)
- Compound 5 450 mg, 1.41 mmol
- 4-methoxyphenylboronic acid 321 mg, 2.12 mmol
- sodium carbonate (448 mg, 4.23 mmol
- distilled water A suspension of (0.7 mL) in DMF (7 mL) was stirred at room temperature for 15 minutes.
- Pd (PPh 3 ) 4 (163 mg, 0.14 mmol) was added to the reaction mixture, and the mixture was stirred at 90 ° C.
- Step F (E) -5- [3- (4-hydroxyphenyl) but-2-en-2-yl] benzene-1,3-diol ((E) -5- [3- (4-hydroxyphenyl) but -2-en-2-yl] benzene-1,3-diol ((E) -5- [3- (4-hydroxyphenyl) but -2-en-2-yl] benzene-1,3-diol) (7)
- dehydrated methylene chloride of compound 6 (2; 1 mixture, 400 mg, 1.34 mmol)
- boron tribromide 1.0 mol / L CH 2 Cl 2 solution, 6.7 mL, 6.7 mmol
- the reaction mixture was separated with distilled water and methylene chloride, and the aqueous layer was further separated with ethyl acetate.
- the obtained organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure.
- Process G 4- [3- (3,5-dimethoxyphenyl) but-2-en-2-yl] -1,2-dimethoxybenzene (4- [3- (3,5-dimethoxyphenyl) but-2-en -2-yl] -1,2-dimethoxybenzene) (8) Compound 5 (1.64 g, 5.15 mmol), 3,4-dimethoxyphenylboronic acid (1.41 g, 7.72 mmol), sodium carbonate (1.64 g, 15.4 mmol) and a suspension of distilled water (5 mL) in DMF (25 mL) was stirred at room temperature for 15 minutes.
- Step H 4- [3- (3,5-dihydroxyphenyl) but-2-en-2-yl] benzene-1,2-diol (4- [3- (3,5-dihydroxyphenyl) but-2-en -2-yl] benzene-1,2-diol) (9)
- a solution of compound (E) -8 (183 mg, 0.56 mmol) in methylene chloride (5.6 mL) was cooled to -20 ° C.
- Boron tribromide 1.0 mol / L CH 2 Cl 2 solution, 3.35 mL, 3.35 mmol
- ESR Electron Spin Resonance
- Resveratrol (R) and the compounds of the present invention, dimethylresveratrol (DR) and dimethylhydroxyresveratrol (DHR) were added to the standard radical.
- the remaining amount of tyrosine radical and hydroxy radical was investigated.
- myoglobin as a tyrosine radical, a standard solution consisting of 50 mM NaPi buffer (pH 7.4), 10 mM DMPO, 1.6 mM myoglobin, and 1 mM H 2 O 2 was prepared.
- Fe 2+ -EDTA was used as the hydroxy radical
- a standard solution consisting of 50 mM NaPi buffer (pH 7.4), 10 mM DMPO, 0.1 mM Fe 2+ -EDTA, and 1 mM H 2 O 2 was prepared.
- 50 mM NaPi buffer (pH 7.4), 10 mM DMPO, 0.1 mM Fe 2+ -EDTA, and 1 mM H 2 O 2 was prepared.
- 5 ⁇ M, 50 ⁇ M and 500 ⁇ M resveratrol (R), dimethylresveratrol (DR) and dimethylhydroxyresveratrol (DHR) were added, and ESR measuring device RX-1 (JEOL Ltd.)
- ESR electron spin resonance
- the ESR settings were a microwave output of 10 mW, an adjustment frequency of 100 KHz, an adjustment magnetic field of 0.1 G, a receiver gain of 1000, and a time constant of 0.3 s.
- the results are shown in FIGS. 1 (A) and 1 (B), respectively.
- each compound suppresses radical generation in a concentration-dependent manner and has strong radical generation inhibitory activity in the order of resveratrol (R) ⁇ dimethylresveratrol (DR) ⁇ dimethylhydroxyresveratrol (DHR).
- R resveratrol
- DR dimethylresveratrol
- DHR dimethylhydroxyresveratrol
- Example 3 Curing efficacy test using smoking rats As described in WO2012 / 161112, a keratoconjunctival epithelial disorder model by dry eye was prepared by the following method, and resveratrol (R), dimethylresveratrol (DR ) And dimethylhydroxyresveratrol (DHR) were evaluated for the healing effect on keratoconjunctival epithelial disorder.
- R resveratrol
- DR dimethylresveratrol
- DHR dimethylhydroxyresveratrol
- mainstream smoke 300 mL was added 6 times every 30 minutes into the chamber containing the rats, and treatment for 12 days caused keratoconjunctival epithelial disorder.
- a PBS solution of 300 ⁇ M resveratrol (R), dimethylresveratrol (DR), or dimethylhydroxyresveratrol (DHR) was each once at 5 ⁇ L, once before smoking treatment and three times after treatment. A total of 4 times a day for 11 days. The animals were 4 (8 eyes) in each group.
- the amount of lacrimal fluid was measured by the cotton thread method for the lacrimal fluid accumulated in the rat cornea. That is, after anesthetizing rats by instilling oxybubrocaine hydrochloride, a cotton thread was inserted between the eyelid and the eyeball for 60 seconds, and tears were collected. The amount of tears was evaluated by measuring the length (mm) of wet cotton yarn.
- the damaged part of the corneal epithelium was stained with the fluorescent dye fluorescein.
- the degree of corneal epithelial damage was determined by dividing the entire cornea into nine parts, upper, middle, lower, and left middle right, and scoring the damage for each part according to the following criteria, and obtaining the total value. Thereafter, the score values were compared between groups. In the statistical analysis, the significant difference of each group with respect to the smoking treatment group was tested using the Dunnett method. In order to make a fair evaluation, from the start of instillation to scoring of corneal epithelial disorder, the contents of the drug solution administered to each group were blinded and collated after scoring.
- the fluorescence staining score increased in the smoking treatment group (fluorescence score average value: 3.7 ⁇ 1.0) compared to the non-smoking treatment group (fluorescence score average value: 0.8 ⁇ 1.0), but decreased due to drug administration (DR Group (fluorescence score average value: 2.3 ⁇ 0.5), p ⁇ 0.05 for smoking treatment group; DHR group (fluorescence score average value: 1.5 ⁇ 0.5), p ⁇ 0.005 for smoking treatment group, ophthalmic solution of the present invention
- DR Group fluorescence score average value: 2.3 ⁇ 0.5
- DHR group fluorescence score average value: 1.5 ⁇ 0.5
- p ⁇ 0.005 for smoking treatment group
- the agent of the present invention is dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, or corneal ulcer It is useful as a preventive and / or therapeutic agent for keratoconjunctival diseases such as
- Lens elasticity is an index of presbyopia because the lens hardens with aging, but increased in the smoking treatment group (elastic modulus: 1.04 ⁇ 0.05) compared to the non-smoking group (elastic modulus: 0.75 ⁇ 0.04).
- R group (elasticity: 0.87 ⁇ 0.06; p ⁇ 0.005 for smoking treatment group; DR group (elasticity: 0.86 ⁇ 0.06); p ⁇ 0.005 for smoking treatment group; DHR group) (Elastic modulus: 0.75 ⁇ 0.05), p ⁇ 0.005 with respect to the smoking treatment group)
- Significant lens hardening was observed, and the lens hardening could be suppressed by instillation of the drug of the present invention (FIG. 5).
- the agent of the present invention is useful as a prophylactic and / or therapeutic agent for presbyopia.
- Example 4 Measurement of radical elimination activity Active oxygen such as hydroxyl radical and peroxyl radical is very reactive with antioxidants and various organic compounds, and does not absorb in the visible region. It is very difficult to perform kinetic analysis on the radical scavenging ability of.
- Galvinoxyl Radical GO ⁇ is a relatively stable organic oxygen radical with a maximum absorption characteristic at 428 nm, so that kinetic analysis of antioxidant radical scavenging ability can be performed by UV-visible spectroscopy instead of active oxygen. Is possible.
- the radical scavenging rate was measured using the stopped flow method.
- the used reagent is as follows. Acetonitrile (Nacalai Tesque, 00433-95), Galvinoxyl Radical (Aldrich, G30-7)
- Measuring method GO / acetonitrile solution (about 2.4 ⁇ 10 ⁇ 6 M) was bubbled with argon for 7 minutes to remove molecular oxygen.
- antioxidants resveratrol, dimethylresveratrol, dimethylhydroxyresveratrol
- the solution was adjusted and molecular oxygen was removed by argon bubbling for 7 minutes.
- GO ⁇ and the acetonitrile solution of the compound were mixed at the same time by the stopped flow method, and the change with time in the absorption maximum (428 nm) of GO ⁇ was measured.
- Equation (4) shows that when ln (A t ⁇ A ⁇ ) is plotted against time t, a straight line is obtained, and the pseudo first-order rate constant k obs is obtained from the slope. In fact, when ln (A t ⁇ A ⁇ ) is plotted against time t, a straight line is obtained, and k obs is determined from this slope. The obtained k obs increased in proportion to the increase in antioxidant concentration.
- the radical scavenging activity of the antioxidants tested in the examples is as follows. Dimethylhydroxyresveratrol was found to have an unexpectedly high radical scavenging activity over resveratrol and dimethylresveratrol. Resveratrol: 4.1 M -1 s -1 Dimethyl resveratrol: 0.96 M -1 s -1 Dimethylhydroxyresveratrol: 2300 M -1 s -1
- Example 5 Curing efficacy test using smoking rats A keratoconjunctival epithelial injury model by dry eye was prepared by the method described in Example 3, and dimethylresveratrol (DR) and 3% diquas, a commercially available dry eye treatment agent, were used. The curative effect of (registered trademark) eye drops (Santen Pharmaceutical Co., Ltd., active ingredient diquafosonal sodium) on keratoconjunctival epithelial disorder was compared.
- DR dimethylresveratrol
- diquas a commercially available dry eye treatment agent
- Smoking treatment was performed on male SD rats (6 weeks old) to prepare a dry eye model.
- mainstream smoke 300 mL was added 6 times every 30 minutes into the chamber containing the rats, and treatment for 12 days caused keratoconjunctival epithelial disorder.
- the animals were 6 non-smoking groups (12 eyes), 4 smoking treatment groups (Smoking), dimethylresveratrol (DR) group, and diquafosol sodium group (4 animals each) 8 eyes).
- the amount of tear fluid and the fluorescence staining score were measured by the methods described in Example 3.
- the fluorescence staining score increased in the smoking treatment group (fluorescence score average value: 4.6 ⁇ 1.2) compared to the non-smoking group (fluorescence score average value: 1.2 ⁇ 1.2), but significantly decreased in the administration of the DR group.
- DR group fluorescence score average value: 2.1 ⁇ 0.8, p ⁇ 0.05 compared to smoking treatment group
- diquafosonal sodium group showed no improvement trend (fluorescence score average value: 4.3 ⁇ 1.6)
- dimethylresveratrol instillation significantly reduced the fluorescence staining score relative to diquafosonal sodium instillation (p ⁇ 0.05).
- the drug dimethylresveratrol of the present invention is compared with diquafosonal sodium, which is a known dry eye treatment drug. It is useful as a preventive and / or therapeutic agent for keratoconjunctival diseases such as dry eye, dry keratoconjunctivitis, punctate superficial keratopathy, corneal erosion, or corneal ulcer.
- Example 6 Safety test of the drug of the present invention
- the rat cornea that had not been smoked or treated with a drug and the cornea of a rat treated with a dimethylresveratrol drug (300 ⁇ M PBS solution) for 1 week were subjected to hematoxylin and eosin staining. went. Photographs of the respective tissue sections are shown in FIGS. 8 (A) and (B).
- Example 7 Stability Test of Drug of the Present Invention Resveratrol and dimethylresveratrol (DR) solid and 10 mM DMSO solution (10% DMSO in water) were allowed to stand at 25 ° C. and 60% relative humidity for a certain period of time. The amount of each subsequent compound was measured by HPLC to evaluate stability. The amount of compound is (Amount of compound after a certain time) / (Amount of compound at the start of the test) ⁇ 100 (%) As calculated. The results are shown in Table 1.
- dimethyl resveratrol had stability comparable to resveratrol.
- dimethylresveratrol showed relatively high stability even after 1 week, and higher stability than resveratrol after 2 weeks.
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Abstract
Description
本願は、2014年8月28日に出願した特願2014-174332号明細書の優先権の利益を主張するものであり、当該明細書はその全体が参照により本明細書中に援用される。
(技術分野)
本発明は、スチルベン化合物を含有する角結膜疾患又は老視の予防及び/又は治療のための医薬組成物に関する。
本発明の目的は、角結膜疾患又は老視の予防及び/又は治療に有効な新規なスチルベン化合物を提供することである。
本発明の別の目的は、強力な抗酸化作用を有する新規なスチルベン化合物を提供することである。
項1.式(I)で表される化合物、その塩又はプロドラッグを有効成分として含有する角結膜疾患又は老視の予防及び/又は治療のための医薬組成物。
式(I)で表される化合物の各芳香環は置換されていてもよい。)
項2.m及びnがそれぞれ1から3までの整数である項1に記載の医薬組成物。
項3.前記化合物が、式(II)で表される化合物、その塩又はプロドラッグである項2に記載の医薬組成物。
項4.前記化合物が、式(III)で表される化合物、その塩又はプロドラッグである項3に記載の医薬組成物。
項5.式(I)で表される化合物、その塩又はプロドラッグを有効成分として含有する角結膜疾患又は老視の予防及び/又は治療剤。
項6.角結膜疾患又は老視を予防及び/又は治療するために使用される式(I)で表される化合物、その塩又はプロドラッグ。
式(I)で表される化合物の各芳香環は置換されていてもよい。)
項7.有効量の式(I)で表される化合物、その塩又はプロドラッグを患者に投与することを含む、角結膜疾患又は老視を予防及び/又は治療するための方法。
式(I)で表される化合物の各芳香環は置換されていてもよい。)
本発明の化合物は以下の式(I)で表される化合物を含む。
まず、エチルフェニルスルホン (EtSO2Ph)のテトラヒドロフラン(THF)溶液を冷却し、ブチルリチウム(BuLi)を滴下、撹拌したものに、芳香環の水酸基をメチル基で保護したメトキシベンズアルデヒド(1)のTHF溶液を反応混合物に滴下し、さらに撹拌する。反応混合物を分液し、有機層を乾燥させる。減圧留去後、残渣を塩化メチレン(CH2Cl2) およびジメチルホルムアミド(DMF)に溶解させ、さらにモレキュラーシーブスおよび二クロム酸ピリジニウム(PDC)を加え撹拌し、反応混合物に酢酸エチルを加え、セライトろ過によりクロム酸塩を除去した後、ろ液を減圧留去する。残渣をカラムクロマトグラフィーにより精製し、式(2)の化合物を得る。
次に、アルゴン雰囲気下にて、塩化テトラブチルアンモニウム(Bu4NCl)及びジエチレングリコールジメチルエーテル(diglyme)の混合物に臭化メチルマグネシウム(MeMgBr)を滴下、撹拌する。反応混合物を冷却した後、式(2)の化合物のTHF溶液を滴下し、さらに撹拌する。反応混合物を分液し、有機層を乾燥させる。減圧留去後、残査をカラムクロマトグラフィーにより精製し、式(3)の化合物を得る。
次に、アルゴン雰囲気下にて、式(3)の化合物の塩化メチレン溶液に、4-N,N-ジメチルアミノピリジン(DMAP) 、N-エチルジイソプロピルアミン(i-Pr2NEt)および無水トリフルオロ酢酸 (F3CCO)2O)を加え撹拌する。反応混合物を分液し、有機層を乾燥させる。減圧留去により得られた残渣を無水トルエンに溶解した後、アルゴン雰囲気下においてジアザビシクロウンデセン(DBU)を加え、さらに撹拌する。反応混合物を分液し、有機層を乾燥させる。減圧留去により得られた残渣をカラムクロマトグラフィーにより精製し、式(4)の化合物を得る。
次に、アルゴン雰囲気下、式(4)の化合物、水素化トリブチルスズ (Bu3SnH)、アゾビスイソブチロニトリル(AIBN)およびN-エチルジイソプロピルアミンのベンゼン溶液を加熱還流させる。反応混合物を分液し、有機層を乾燥させる。減圧留去により得られた残渣を四塩化炭素 (CCl4)に溶解させた後、アルゴン雰囲気下においてヨウ素を加え撹拌する。反応混合物を分液し、有機層を減圧留去する。得られた残渣をさらに分液し、有機層を乾燥させる。減圧留去により得られた残渣をカラムクロマトグラフィーにより精製し、式(5)の化合物を得る。
次に、式(5)の化合物、メトキシ(メチル基は1又は複数)フェニルボロン酸 、炭酸ナトリウム および蒸留水 のDMF の懸濁液を撹拌する。反応混合物にPd(PPh3)4を加え、加熱撹拌する。反応混合物を分液し、有機層を乾燥させた後、減圧留去する。残渣をカラムクロマトグラフィーにより精製し、式(6)の化合物をシス体とトランス体との混合物として得る。なお、このシス体とトランス体はHPLCにより分離可能である。
次に、アルゴン雰囲気下において、式(6)の化合物 (シス体とトランス体との混合物) の脱水塩化メチレン溶液に三臭化ホウ素 (BBr3)を滴下する。反応混合物を撹拌した後、さらに撹拌する。反応混合物を分液し、水層をさらに別の溶媒で分液する。得られた有機層を乾燥した後、減圧留去する。残渣をカラムクロマトグラフィーにより粗精製し、式(7)の化合物を混合物として得る。
レスべラトロールはSigma-Aldrich社より入手した(製品番号R5010-100MG)。本発明の式(V)で表される化合物である (E)-5-[3-(4-ヒドロキシフェニル)ブタ-2-エン-2-イル]ベンゼン-1,3-ジオール(DR、以下「ジメチルレスベラトロール」と称する)及び式(VI)で表される化合物である(E)-4-[3-(3,5-ジヒドロキシフェニル)ブタ-2-エン-2-イル]ベンゼン-1,2-ジオール(DHR、以下「ジメチルヒドロキシレスベラトロール」と称する)を下記のスキームに従って製造した。
エチルフェニルスルホン(EtSO2Ph) (7.72 g, 45.1 mmol) のTHF (60 mL) 溶液を-80 ℃に冷却し、ブチルリチウム (2.66 mol/L n-ヘキサン(以下、単に「ヘキサン」とする)溶液, 17.0 mL, 45.1 mmol) を滴下した後40分間撹拌した。3,5-ジメトキシベンズアルデヒド (1) (5.0 g, 30.09 mmol) のTHF (30 mL) 溶液を反応混合物に滴下し、さらに30分間撹拌した。反応混合物を飽和塩化アンモニウム水溶液および酢酸エチルで分液し、有機層を無水硫酸ナトリウムで乾燥させた。減圧留去の後、得られた残渣を塩化メチレン (50 mL) およびDMF (10 mL)に溶解させた。さらにモレキュラーシーブス4Å (10 g) およびPDC (56.6 g, 150.5 mmol) を加え、室温にて18時間撹拌した。反応混合物に酢酸エチル (50 mL)を加えた後、セライトろ過によりクロム酸塩を除去した後、ろ液を減圧留去した。残渣をカラムクロマトグラフィー(シリカゲル、ヘキサン/塩化メチレン/酢酸エチル = 20/20/1(体積比))により精製し、化合物2 (8.16 g, 81%)を白色固体として得た。
1H NMR (400 MHz, CDCl3) 0. 1.57 (d, J = 7.2 Hz, 3H), 3.83 (s, 6H), 6.67-6.68 (m, 1H), 7.07 (d, J = 2.0 Hz, 2H), 7.51-7.55 (m, 2H), 7.64-7.68 (m, 1H), 7.80-7.82 (m, 2H); 13C NMR (125 MHz, CDCl3) 13.3, 55.6, 65.1, 106.4, 106.8, 128.9, 129.8, 134.2, 136.1, 138.0, 160.9; FAB-MS m/z 335 (M++H).
アルゴン雰囲気下0℃において、塩化テトラブチルアンモニウム (167 mg, 0.6 mmol) およびジエチレングリコールジメチルエーテル (1.28 mL, 9.0 mmol) の混合物に臭化メチルマグネシウム (1.0 mol/L THF溶液, 9.0 mL, 9.0 mmol) を滴下し30分間撹拌した。反応混合物を-40℃に冷却した後、化合物2 (1.0 g, 3.0 mmol) のTHF (15 mL) 溶液を滴下しさらに25時間撹拌した。反応混合物を飽和塩化アンモニウム水溶液および酢酸エチルで分液し、有機層を無水硫酸ナトリウムにより乾燥させた。減圧留去の後、残渣をカラムクロマトグラフィー(シリカゲル、ヘキサン/塩化メチレン/酢酸エチル = 15/15/1(体積比))により精製し、化合物3 (661 mg, 63%)を無色アメ状物質として得た。
1H NMR (400 MHz, CDCl3) 0.96 (d, J = 7.2 Hz, 3H), 1.92 (s, 3H), 3.50 (q, J = 7.2 Hz, 1H), 3.79 (s, 6H), 3.95 (s, 1H), 6.34 (t, J = 2.4 Hz, 1H), 6.57 (d, J = 2.4 Hz, 2H), 7.54-7.59 (m, 2H), 7.63-7.67 (m, 1H), 7.85-7.88 (m, 2H); 13C NMR (125 MHz, CDCl3) 12.4, 30.8, 55.4, 67.7, 75.7, 98.7, 103.1128.3, 129.2, 133.8, 139.1, 148.0, 160.7; FAB-MS m/z 351 (M++H).
アルゴン雰囲気下0℃において、化合物3 (1.71 g, 4.89 mmol) の塩化メチレン (40 mL)溶液に、4-N,N-ジメチルアミノピリジン (597 mg, 4.89 mmol)、N-エチルジイソプロピルアミン (1.7 mL, 9.77mmol)および無水トリフルオロ酢酸 (1.38 mL, 9.77 mmol) を加え、室温にて90分間撹拌した。反応混合物を飽和炭酸水素ナトリウム水溶液および塩化メチレンにより分液し、有機層を無水硫酸ナトリウムにより乾燥させた。減圧留去により得られた残渣を無水トルエン (40 mL) に溶解した後、アルゴン雰囲気下0℃においてジアザビシクロウンデセン(DBU) (2.92 mL, 19.5 mmol) を加え、室温にてさらに80分間撹拌した。反応混合物を0.5規定塩酸および酢酸エチルで分液し、有機層を無水硫酸ナトリウムにより乾燥させた。減圧留去により得られた残渣をカラムクロマトグラフィー(シリカゲル、ヘキサン/酢酸エチル = 3/1(体積比))により精製し、化合物4 (990 mg, 61%) を白色固体として得た。
1H NMR (400 MHz, CDCl3) 1.6-1.87 (m, 3H), 2.44-2.45 (m, 3H), 3.77 (s, 6H), 6.19 (d, J = 2.0 Hz, 2H), 6.38 (t, J = 2.0 Hz, 1H), 7.55-7.59 (m, 2H), 7.62-7.64 (m, 1H), 7.94-7.96 (m, 2H); 13C NMR (125 MHz, CDCl3) 17.7, 22.4, 55.4, 99.2, 104.5, 127.2, 129.1, 133.1, 133.7, 141.4, 144.9, 149.7, 161.0; FAB-MS m/z 333 (M++H).
アルゴン雰囲気下、化合物4 (580 mg, 1.75 mmol), 水素化トリブチルスズ (1.41 mL, 5.24 mmol)、アゾビスイソブチロニトリル(AIBN)(143 mg, 0.87 mmol)およびN-エチルジイソプロピルアミン (0.91 mL, 5.24 mmol) のベンゼン (17 mL) 溶液を20時間加熱還流させた。反応混合物を飽和炭酸水素ナトリウム水溶液およびヘキサンで分液し、有機層を無水硫酸ナトリウムにより乾燥させた。減圧留去により得られた残渣を四塩化炭素 (17 mL)に溶解させた後、アルゴン雰囲気下0℃においてヨウ素 (1.77 g, 6.98 mmol) を加え、室温にて90分間撹拌した。反応混合物を飽和炭酸水素ナトリウム水溶液 (100 mL)、飽和チオ硫酸ナトリウム水溶液 (100 mL) および塩化メチレンで分液し、有機層を減圧留去した。得られた残渣をさらに10%フッ化カリウム水溶液および塩化メチレンで分液し、有機層を無水硫酸ナトリウムにより乾燥させた。減圧留去により得られた残渣をカラムクロマトグラフィー(シリカゲル、ヘキサン/酢酸エチル = 20/1(体積比))により精製し、化合物5 (533 mg, 96%, 2;1の混合物) を油状物質として得た。
1H NMR (400 MHz, CDCl3) 2.07-2.08 (m, 1H), 2.21-2.22 (m, 2H), 2.42-2.43 (m, 2H), 2.63-2.64 (m, 1H), 3.78 (s, 4H), 3.80 (s, 2H), 6.27-6.28 (m, 2H), 6.36-6.38 (m, 1H); 13C NMR (125 MHz, CDCl3) 20.5, 30.6, 31.7, 55.3, 94.5, 98.1, 98.8, 98.9, 105.7, 141.8, 142.1, 142.9, 150.3, 160.5, 160.6; FAB-MS m/z 319 (M++H).
化合物5 (450 mg, 1.41 mmol)、4-メトキシフェニルボロン酸 (321 mg, 2.12 mmol)、炭酸ナトリウム (448 mg, 4.23 mmol) および蒸留水 (0.7 mL) のDMF (7 mL) の懸濁液を室温にて15分間撹拌した。反応混合物にPd(PPh3)4 (163 mg, 0.14 mmol) を加え、90℃にて90分間撹拌した。反応混合物を飽和食塩水および酢酸エチルで分液し、有機層を無水硫酸ナトリウムにより乾燥させた後減圧留去した。残渣をカラムクロマトグラフィー(シリカゲル、ヘキサン/酢酸エチル = 30/1(体積比))により精製し、化合物6 (316 mg, 75%、固体) を2:1の混合物として得た。HPLC (カラム:SHIMADZU Shim Pack PRC-SIL (250 × 20 mm), 測定波長: 254 nm, 移動相:ヘキサン/酢酸エチル = 30/1(体積比)、流速:20 mL/min) により、それぞれ (E)-6 (Rt = 5.6 min) および (Z)-6 (Rt = 6.4 min)を分離し機器分析用のサンプルとした。
(E)-6; 1H NMR (400 MHz, CDCl3) 1.88 (s, 6H), 3.82 (s, 6H), 3.83 (s, 3H), 6.37 (t, J = 2.0 Hz, 1H), 6.41 (d, J = 2.0 Hz, 2H), 6.89-6.93 (m, 2H), 7.18-7.21 (m, 2H); 13C NMR (125 MHz, CDCl3) 22.4, 22.5, 55.2, 55.3, 98.2, 106.3, 113.5, 129.3, 132.4, 132.8, 136.5, 146.9, 158.0, 160.6; FAB-MS m/z 299 (M++H).(Z)-6; 1H NMR (400 MHz, CDCl3) 2.13 (s, 6H), 3.58 (s, 6H), 3.72 (s, 3H), 6.13 (d, J = 2.4 Hz, 2H), 6.16 (t, J = 2.4 Hz, 1H), 6.64-6.66 (m, 2H), 6.90-6.93 (m, 2H); 13C NMR (125 MHz, CDCl3) 21.3, 21.5, 55.1, 55.1, 98.0, 107.4, 113.0, 129.9, 132.2, 132.5, 137.1, 146.8, 157.5, 159.9; FAB-MS m/z 299 (M++H).
アルゴン雰囲気下-20℃において、化合物 6 (2;1の混合物、400 mg, 1.34 mmol) の脱水塩化メチレン (10 mL) 溶液に三臭化ホウ素 (1.0 mol/L CH2Cl2溶液, 6.7 mL, 6.7 mmol) を滴下した。反応混合物を-20℃で95分間撹拌した後、室温でさらに95分間撹拌した。反応混合物を蒸留水および塩化メチレンで分液し、水層をさらに酢酸エチルで分液した。得られた有機層を無水硫酸ナトリウムにより乾燥させた後減圧留去した。残渣をカラムクロマトグラフィー(シリカゲル、ヘキサン/酢酸エチル = 1/1(体積比))により粗精製し、化合物7 (330 mg) を混合物として得た。
1H NMR (400 MHz, CD3OD) 1.79 (s, 3H), 1.82 (s, 3H), 6.15-6.16 (m, 3H), 6.76 (d, J = 8.6 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H); 13C NMR (125 MHz, CD3OD) 22.7, 22.9, 101.5, 107.7, 115.9, 130.3, 133.4, 134.0, 136.9, 148.3, 156.8, 159.3; HRMS (FAB+): calcd for C16H17O3 257.1178, Found 257.1168 [M++H].
化合物5 (1.64 g, 5.15 mmol)、3,4-ジメトキシフェニルボロン酸 (1.41 g, 7.72mmol)、炭酸ナトリウム (1.64 g, 15.4 mmol) および蒸留水 (5 mL) のDMF (25 mL) の懸濁液を室温にて15分間撹拌した。反応混合物にPd(PPh3)4 (595 mg, 0.52 mmol) を加え、90℃にて19時間撹拌した。反応混合物を飽和食塩水および酢酸エチルで分液し、有機層を無水硫酸ナトリウムにより乾燥させた後減圧留去した。残渣をカラムクロマトグラフィー(シリカゲル)により精製し、化合物 (E)-8 (ヘキサン/塩化メチレン=2/1(体積比)、870 mg, 52%、固体) および化合物 (Z)-8 (ヘキサン/塩化メチレン/酢酸エチル=20/20/1(体積比)、388 mg, 23%、固体)をそれぞれ得た。
(E)-8; 1H NMR (400 MHz, CDCl3) 1.89 (s, 6H), 3.82 (s, 6H), 3.91 (s, 3H), 3.91 (s, 3H), 6.38 (t, J = 2.4 Hz, 1H), 6.42 (d, J = 2.4 Hz, 2H), 6.79-6.83 (m, 2H), 6.88 (d, J = 8.0 Hz, 1H); 13C NMR (125 MHz, CDCl3) 22.4, 22.6, 55.3, 55.9, 98.2, 106.3, 110.8, 111.6, 120.4, 132.6, 132.9, 136.9, 146.8, 147.4, 148.5, 160.6; FAB-MS m/z 329 (M++H).
(Z)-8; 1H NMR (400 MHz, CDCl3) 2.14 (s, 3H), 2.15 (s, 3H), 3.58 (s, 3H), 3.59 (s, 6H), 3.80 (s, 3H), 6.15-6.17 (m, 3H), 6.47 (d, J = 2.0 Hz), 6.63 (dd, J = 8.0 and 2.0 Hz, 1H), 6.67 (d, J = 8.0 Hz, 1H); 13C NMR (125 MHz, CDCl3) 21.2, 21.3, 55.1, 55.6, 55.7, 97.9, 107.3, 1103, 113.0, 120.7, 132.4, 132.4, 137.2, 146.9, 147.0, 147.8, 160.1; FAB-MS m/z 329 (M++H).
アルゴン雰囲気下、化合物 (E)-8 (183 mg, 0.56 mmol) の塩化メチレン(5.6 mL)溶液を-20 ℃に冷却し、三臭化ホウ素 (1.0 mol/L CH2Cl2溶液, 3.35 mL, 3.35 mmol) を滴下し80分間撹拌した。室温にてさらに90分間撹拌した後、蒸留水 (50 mL)を加えた。反応混合物を塩化メチレンにより抽出した後、さらに水層を酢酸エチルにより抽出した。得られた有機層を合わせて減圧留去した。残渣をHPLC (カラム:SHIMADZU Shim Pack PRC-SIL (250 × 20 mm), 測定波長: 254 nm, 移動相:ヘキサン/酢酸エチル = 1/2(体積比)、流速:20 mL/min)により生成し、化合物9 (Rt = 4.2 min, 55 mg, 36%) を固体として得た。1H NMR (400 MHz, CD3OD) 1.80 (s, 3H), 2.00 (s, 3H), 6.14 (br-s, 3H), 6.53 (dd, J = 8.0 and 2.4 Hz, 1H), 6.65 (d, J = 2.4 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H); 13C NMR (125 MHz, CD3OD) 20.9, 22.9, 101.4, 107.7, 116.1, 116.5, 120.7, 133.6, 133.8, 137.6, 144.7, 145.9, 159.3; FAB-MS m/z 273 (M++H). HRMS (FAB+): calcd for C16H17O4 273.1127, Found 273.1084 [M++H].
標準物質であるラジカルに対し、レスベラトロール(R)並びに本発明の化合物であるジメチルレスベラトロール(DR)及びジメチルヒドロキシレスベラトロール(DHR)を加えたときのチロシンラジカル及びヒドロキシラジカルの残存量を調べた。チロシンラジカルとしてミオグロビンを使用し、50mM NaPi緩衝液(pH7.4)、10mM DMPO、1.6mM ミオグロビン、及び1mM H2O2からなる標準溶液を調製した。また、ヒドロキシラジカルとしてFe2+-EDTAを使用し、50mM NaPi緩衝液(pH7.4)、10mM DMPO、0.1mM Fe2+-EDTA、及び1mM H2O2からなる標準溶液を調製した。これらの標準溶液に対し、5μM、50μM、500μMのレスベラトロール(R)、ジメチルレスベラトロール(DR)及びジメチルヒドロキシレスベラトロール(DHR)を加え、ESR測定装置RX-1(日本電子株式会社製)を用いて電子スピン共鳴(ESR)にてチロシンラジカルの量及びヒドロキシラジカルの量を吸収スペクトルの変化に対し測定した。ESRの設定はマイクロ波出力 10 mW、 調整周波数100 KHz、調整磁場 0.1 G、受信器利得1000、及び時定数0.3 sとした。結果をそれぞれ図1(A)及び図1(B)に示す。
WO2012/161112に記載された通り、以下に示す方法でドライアイによる角結膜上皮障害モデルを作製し、レスベラトロール(R)、ジメチルレスベラトロール(DR)及びジメチルヒドロキシレスベラトロール(DHR)の角結膜上皮障害に対する治癒効果を評価した。
(実験方法)
雄性SDラット(6週齢)に喫煙処理を施し、ドライアイ及び老視モデルを作製した。
(角膜上皮のフルオレセイン染色スコア判断基準)
0:染色されない(点状蛍光なし)
1:わずかに点状蛍光がみられる
2:比較的多く点状蛍光がみられる
3:密に点状蛍光がみられる
水晶体硬度は電子天秤とハイトゲージを組み合わせて測定した。あらかじめ短軸長を測定した水晶体を電子天秤の上に置き、重さを0に合わせた。水晶体の上からハイトゲージのハンドルを操作して、先端部分が水晶体に接するようにした。さらにハンドルを操作して短軸長の5-10%程度先端を下げ、水晶体に圧力をかけた。この時の重さの変化を電子天秤で測定し、重さをハイトゲージに示された移動距離で割り硬度とした。値が大きいほど硬いことを示す。
(結果)
体重は、有意差はないものの、喫煙処理群(Smoking)では喫煙非処理群(Non smoking)に比べて低下傾向にあった(図2)(喫煙非処理群及び喫煙処理群でそれぞれ308.2±10.4g及び285.9±12.2 g)。
ヒドロキシルラジカルやペルオキシルラジカル等の活性酸素は抗酸化物質および様々な有機化合物に対して反応性が非常に高く、また可視部に吸収を持たないため、抗酸化物質のラジカル消去能について速度論的解析を行うことは非常に困難である。一方、Galvinoxyl Radical(GO・)は428nmに特徴的な極大吸収を持つ比較的安定な有機酸素ラジカルであるため、活性酸素に変わって紫外可視分光法で抗酸化ラジカル消去能の速度論的解析が可能である。そこで、GO・と抗酸化物質であるレスベラトロール(R)、ジメチルレスベラトロール(DR)又はジメチルヒドロキシレスベラトロール(DHR)との反応で減少する428nmの変化を速度論的解析することで、これらの抗酸化物質のラジカル消去活性を求めた。ラジカル消去活性の測定方法については公知文献(K. Fukuhara, et al., Chem. Res. Toxicol, 21, 282-287(2008); K. Imai, et al., Bioorg. Med. Chem. Lett., 24, 2582-2584(2014))に記載されている。
本実験はstopped flow法を用いてラジカル消去速度の測定を行った。また、使用した試薬は以下の通りである。
アセトニトリル(ナカライテスク,00433-95),
Galvinoxyl Radical(Aldrich,G30-7)
GO・のアセトニトリル溶液(約2.4×10-6M)を7分間アルゴンバブリングし、分子状酸素を除去した。また、抗酸化物質(レスベラトロール、ジメチルレスベラトロール、ジメチルヒドロキシレスベラトロール)をアセトニトリルに溶解し、2.4×10-5M~2.5×10-3Mの範囲で5点以上の濃度の異なる溶液を調整し、7分間アルゴンバブリングして分子状酸素を除去した。次にstopped flow法により、GO・と化合物のアセトニトリル溶液を同時に混ぜた後、GO・の吸収極大(428nm)の経時変化を測定した。
GO・に由来する428nmの吸光度の減少は抗酸化物質の濃度がGO・の濃度の10倍以上の条件下([抗酸化剤]) > 10[GO・])では抗酸化剤の濃度([抗酸化物質])に対して擬一次速度式に従う(方程式(1))。
-d[GO・] / dt = kobs[GO・] (1)
ここでkobsは擬一次速度定数である。方程式(1)の両辺を積分すると方程式(2)が得られる。
ln([GO・] / [GO・]0) = - kobs t (2)
ここで[GO・]0はt=0のときのGO・の濃度である。
[GO・] / [GO・]0 = (At - A∞) / (A0 - A∞) (3)
ln(At - A∞) = - kobst + ln(A0 - A∞) (4)
方程式(4)は、ln(At - A∞)を時間tに対してプロットすると、直線が得られ、その傾きから擬一次速度定数kobsが求められることを示している。実際、ln(At - A∞)を時間tに対してプロットすると直線が得られ、この傾きからkobsを決定した。得られたkobsは抗酸化物質の濃度の増加に比例して増加した。
kobs= kHT [抗酸化物質] (5)
以上より全体の反応速度は方程式(6)で表され、GO・と抗酸化剤のそれぞれの濃度に対して一次反応であることがわかった。
Rate = d[GO・] / dt = kHT[GO・][抗酸化物質] (6)
kobsを縦軸に、[抗酸化物質]を横軸に取ったグラフの直線の傾き(GO・への水素移動速度定数(kHT))が各抗酸化物質のラジカル消去活性であり、本実施例で試験した抗酸化物質のラジカル消去活性は以下の通りである。ジメチルヒドロキシレスベラトロールはレスベラトロール及びジメチルレスベラトロールに対し、予想外の高いラジカル消去活性を有することが分かった。
レスベラトロール:4.1 M-1s-1
ジメチルレスベラトロール:0.96 M-1s-1
ジメチルヒドロキシレスベラトロール:2300 M-1s-1
実施例3に記載した方法でドライアイによる角結膜上皮障害モデルを作製し、ジメチルレスベラトロール(DR)及び市販のドライアイ治療剤である3%ジクアス(登録商標)点眼液(参天製薬株式会社、有効成分 ジクアホソナルナトリウム)の、角結膜上皮障害に対する治癒効果を比較した。
(結果)
体重は、喫煙非処理群(Non smoking)、喫煙処理群(Smoking)、ジメチルレスベラトロール群、及びジクアホソナルナトリウム群で有意差はなかった(喫煙非処理群、喫煙処理群、DR群、及びジクアホソナルナトリウム群でそれぞれ281.6±7.4g、280.0±5.8 g、271.0±7.5g、及び273.8±4.3g)。
喫煙処理も薬剤による処理も施していないラットの角膜及びジメチルレスベラトロール薬剤(300μM PBS溶液)で1週間処理したラットの角膜について、ヘマトキシリン・エオジン染色を行った。それぞれの組織切片の写真を図8(A)及び(B)に示す。
レスべラトール及びジメチルレスベラトロール(DR)の固体および10mM DMSO溶液(10% DMSO in water) を、25℃、60%相対湿度で放置し、一定時間後のそれぞれの化合物の量をHPLCで測定して安定性を評価した。化合物の量は、
(一定時間後の化合物の量)/(試験開始時の化合物の量)×100(%)
として計算した。結果を表1に示す。
Claims (5)
- m及びnがそれぞれ1から3までの整数である請求項1に記載の医薬組成物。
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WO2007020673A1 (en) * | 2005-08-19 | 2007-02-22 | Tubilux Pharma S.P.A. | Use of glucosylated hydroxystilbenes for the prevention and treatment of eye pathologies |
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